The development of a novel SNP genotyping assay to differentiate cacao clones

In this study, a double-mismatch allele-specific (DMAS) qPCR SNP genotyping method has been designed, tested and validated specifically for cacao, using 65 well annotated international cacao reference accessions retrieved from the Center for Forestry Research and Technology Transfer (CEFORTT) and the International Cocoa Quarantine Centre (ICQC). In total, 42 DMAS-qPCR SNP genotyping assays have been validated, with a 98.05% overall efficiency in calling the correct genotype. In addition, the test allowed for the identification of 15.38% off-types and two duplicates, highlighting the problem of mislabeling in cacao collections and the need for conclusive genotyping assays. The developed method showed on average a high genetic diversity (He = 0.416) and information index (I = 0.601), making it applicable to assess intra-population variation. Furthermore, only the 13 most informative markers were needed to achieve maximum differentiation. This simple, effective method provides robust and accurate genotypic data which allows for more efficient resource management (e.g. tackling mislabeling, conserving valuable genetic material, parentage analysis, genetic diversity studies), thus contributing to an increased knowledge on the genetic background of cacao worldwide. Notably, the described method can easily be integrated in other laboratories for a wide range of objectives and organisms

A combined RNA preservation and extraction protocol for gene expression studies in cacao beans

Despite the high economic importance of cacao beans, few RNA-based studies have been conducted on this plant material and hence no optimal RNA-extraction has been reported. Moreover, extraction of high-quality RNA from recalcitrant cacao bean tissue has shown many difficulties and requires optimization. Furthermore, cacao beans are mostly found at remote and under-resourced locations, which pressures the outsourcing of such analysis and thereby demands RNA-stable preservation and transportation of cacao beans. This study aims to select an appropriate RNA extraction and preservation/transportation method for cacao beans. For this purpose, three sample homogenization and five extraction protocols on cacao beans were compared. In addition, 13 preservation conditions-differing in tissue crushing degree, preservation method, duration, and temperature-were compared and evaluated. A comparative analysis revealed that CTAB-based homogenization and extraction outcompeted all tested commercial protocols in RNA yield and integrity, respectively. Preservation at -80 degrees C affected RNA quality the least, whereas freeze-drying was most suitable for transportation at room temperature for maximum 1 week. The cacao bean RNA obtained from the selected methods were compatible for downstream applications. The results of this study will facilitate on-field sampling and transportation of genetically sensitive cacao material prior to cacao bean transcriptomic studies. In addition, valuable insights on sample homogenization, extraction, preservation, and transportation have been provided, which is of interest to every plant geneticist

Discrimination of cocoa liquors based on their odor fingerprint : a fast GC electronic nose suitability study

With the rising interest by consumers for high-quality cocoa products from a clear geographical origin, a rapid analytical method for quality control, authenticity and traceability assessment is of paramount importance. However, the complex mixture of volatiles present in cocoa liquor, the main ingredient for the chocolate production, complicates reaching this purpose. Hence, an analytical fingerprint approach using advanced electronic nose (E-nose) technology may offer a suitable technique. This study aimed to verify the suitability of an E-nose based on ultra-fast gas chromatography (GC) for the rapid discrimination between cocoa liquors from different origins. Fourteen cocoa liquors, produced of cocoa beans from ten different geographical origins, were analyzed. The obtained odor fingerprints were investigated using principal component analysis (PCA) which successfully discriminated most cocoa liquors, within one continent, according to their geographical origin. Besides, discriminant factor analysis (DFA) showed the possibility to differentiate between bulk and fine cocoa. Further tentative identification of predominant volatile compounds allowed the detection of compounds within a wide range of chemical classes occurring in cocoa products, such as acids, alcohols, aldehydes, ketones, esters, pyrazines, pyrones, and pyrroles. Most odorant compounds were previously described in literature as key volatiles in cocoa flavor, notable examples are acetic acid, 2-heptanol, 2/3-methylbutanal, acetophenone, isoamyl acetate, tetramethylpyrazine, maltol, and 2-acetyl-1-pyrroline. This study proves for the first time the usefulness of the GC E-nose for effective and rapid aroma profiling and discrimination between single origin cocoa liquors, which can be easily applied in the cocoa industry

Dynamics of volatile compounds and flavor precursors during spontaneous fermentation of fine flavor Trinitario cocoa beans

Fine flavor cocoa is worldwide renowned to produce origin chocolates with special aromas, e.g. fruity or floral, in addition to its chocolate aroma. This research aims to elucidate fine flavor dynamics during fermentation by analyzing the sugar, free amino acid (FAA) and volatile profile. Ecuadorian Trinitario beans (Sacha Gold) were sampled after 0, 18, 24, 48 and 66 h of spontaneous fermentation. The unfermented beans contained significant sucrose, glutamic acid and asparagine amounts while the fermented beans (66 h) contained more flavor precursors, e.g. glucose, fructose, hydrophobic and other FAA. Forty-one volatiles were identified, including 13 fruity- and 12 floral-like, derived from various metabolic pathways. Whereas the level of fatty acid-derived fruity volatiles decreased, the amount of amino acid-derived fruity and floral volatiles increased and floral terpenes remained stable. Some fine volatiles were assumed to be pulp-derived (e.g. linalool, beta-myrcene, 2-heptyl acetate) or intrinsic to the bean (e.g. 2-heptanol, 2-heptanone, 2-pentanol), while others were generated during fermentation by microbial synthesis (e.g. 2-phenylethanol, isoamyl alcohol). Multivariate analysis clustered samples according to fermentation time and quality. These findings demonstrate that cocoa fermentation is essential for the formation of flavor precursors and the development or preservation of important fine aroma compounds. Trinitario (or hybrids), one of the cocoa varieties with fine flavor potential, is cultivated all over the world and hence, care should be taken during post-harvest to fully exploit this fine flavor character and deliver high-quality cocoa beans with fine sensory characteristics

Dynamics of volatile compounds and flavor precursors during spontaneous fermentation of fine flavor Trinitario cocoa beans

Fine flavor cocoa is worldwide renowned to produce origin chocolates with special aromas, e.g. fruity or floral, in addition to its chocolate aroma. This research aims to elucidate fine flavor dynamics during fermentation by analyzing the sugar, free amino acid (FAA) and volatile profile. Ecuadorian Trinitario beans (Sacha Gold) were sampled after 0, 18, 24, 48 and 66 h of spontaneous fermentation. The unfermented beans contained significant sucrose, glutamic acid and asparagine amounts while the fermented beans (66 h) contained more flavor precursors, e.g. glucose, fructose, hydrophobic and other FAA. Forty-one volatiles were identified, including 13 fruity- and 12 floral-like, derived from various metabolic pathways. Whereas the level of fatty acid-derived fruity volatiles decreased, the amount of amino acid-derived fruity and floral volatiles increased and floral terpenes remained stable. Some fine volatiles were assumed to be pulp-derived (e.g. linalool, β-myrcene, 2-heptyl acetate) or intrinsic to the bean (e.g. 2-heptanol, 2-heptanone, 2-pentanol), while others were generated during fermentation by microbial synthesis (e.g. 2-phenylethanol, isoamyl alcohol). Multivariate analysis clustered samples according to fermentation time and quality. These findings demonstrate that cocoa fermentation is essential for the formation of flavor precursors and the development or preservation of important fine aroma compounds. Trinitario (or hybrids), one of the cocoa varieties with fine flavor potential, is cultivated all over the world and hence, care should be taken during post-harvest to fully exploit this fine flavor character and deliver high-quality cocoa beans with fine sensory characteristics.status: publishe

Unraveling the genetic background of the Yangambi Research Center cacao germplasm collection, DR Congo

The Democratic Republic of the Congo (DR Congo or DRC) has some of the most fertile soils in Africa to cultivate cacao, an important cash crop and source of income for many smallholder farmers. Although cacao is currently produced there on small scale, DRC has the potential to grow as a cacao-producing country, thereby supplying the increasing cacao demand in the global market. Since the introduction of cacao varieties in the late nineteenth century, selection and breeding experiments have been carried out based on phenotype, without any knowledge on the genetic background of the cultivars. Therefore, this study analyzes 62 Congolese accessions of the Centre de Recherche de Yangambi (CRY) and 51 accessions of international collections representing 10 reference groups and Trinitario cultivars using 14 microsatellite markers. Descriptive statistics revealed a high gene diversity and polymorphic information content (PIC) for most of the markers, of which mTcCIR 12, 37, and 60 were the most discriminative. Both Bayesian clustering and principal coordinate analysis (PCoA) revealed high-admixed ancestry of the CRY cultivars. The collection was divided in two clusters, of which the first formed a hybrid population, linked to Amelonado, Trinitario, MaraIOEn, and Nanay, and the second was assigned to predominantly Amelonado, followed by Nanay, Contamana, and Nacional. This high admixture level resulted from numerous hybridization and recombination events that took place in the previous century. The obtained knowledge is essential for efficient conservation, utilization, and selection of high-quality cacao cultivars, which are agronomical favored, disease resistant, and of superior flavor quality

A multipronged flavor comparison of Ecuadorian CCN51 and Nacional cocoa cultivars

Two cocoa types are predominantly being cultivated in Ecuador, the highly appreciated fine Nacional and the high-yielding and disease-resistant CCN51, classified as bulk. With the introduction of foreign germplasm in Ecuador and the international demand for high-quality Nacional cocoa, the present study aims to characterize and compare the flavor profile of Nacional hybrids (EET103, EET559, EET576 and EET577) and CCN51, and to assess the effect of the genotype. The fermented dried beans were processed into liquor and their sensory properties, volatile composition and odor fingerprint were analyzed by trained panelists, headspace-solid phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS), and GC electronic nose (e-nose), respectively, combined with multivariate statistics. Seventy volatiles were identified and semi-quantified, among them, 16 and 14 volatiles conferring fruity and floral aroma notes, respectively. CCN51 was mainly characterized by undesirable odor-active volatiles, whereas the EET cultivars contained the highest concentration of most fruity and floral volatiles, with differences in between EET cultivars. Sensory data highlighted organoleptic differences between the fine and bulk liquors, i.e. the fruity and acid EET flavor strongly differed from the bitter, astringent and off-flavor attributes of CCN51. Principal component analysis (PCA) discriminated firstly between fine and bulk, and secondly between EET identity. Partial least squares analysis identified possible correlations between sensory attributes and volatile compounds. Besides, e-nose proved its usefulness for rapid assessment of the liquors' sensory profile. The results provide a foundation for understanding the genotype's contribution to and the compositional basis of Ecuadorian fine and bulk cocoa flavor